This lab focuses on developing and analyzing animal models of retinal degeneration toward developing human treatment. Analysis includes retinal structure and functional responses using the electroretinogram (ERG). This year we focused on four areas: (1) Analysis of a retinoschisis knockout we developed and of an ELOVL4 knockout mouse. (2) Functional screening of other murine models to explore the possible role of these genes in normal and diseased retina. (3) Mechanisms producing functional ERG changes in animal models and human retinal degeneration. (4) Investigated possible treatments in animal models, including mechanisms and possible adverse effects. Juvenile X-linked retinoschisis(XLRS): RS-1 gene mutations cause XLRS, in which the inner retinal layers delaminate. We evaluated local cellular expression of RS-1 in mouse and found a spatial-temporal 'wave' of retinoschisin expression during retinal development and by all major classes of adult retinal neurons, suggesting that retinoschisin in the inner retina is synthesized locally rather than being transported from distal retinal photoreceptors as earlier proposed. RS expression by adult neurons supports possible future therapy by protein replacement to treat infants and (surprisingly) adults with XLRS. Gene transfer therapy for retinoschisis in the murine RS-1 knockout model: We created an RS1 knockout mouse and investigated whether RS1 gene delivery can reverse the abnormal 'electronegative' ERG of retinoschisis knockout (Rs1h-KO) mouse which mimics human XLRS disease and shows abnormal synaptic transfer in the absence of protein. Intraocular administration of AAV(2/2)-CMV-Rs1h caused retinoschisin expression in all retinal layers of Rs1h_/Y mice and restored the normal ERG waveform, indicating that gene therapy is a viable strategy of therapeutic intervention even in postdevelopmental adult XLRS disease. ELOVL4 knockout mice: ELOVL4 mutations cause human Stargardt-like macular dystrophy. Characterization of the ELOVL4 knockout mouse should help confirm or refute its involvement in biosynthesis of essential fatty acids. We are evaluating the retinal fatty acid content, functional responses and histopathology of this model and plan a treatment study using dietary supplementation with fatty acids. Retinal Light Damage: Damage to the retina of rodents by moderate levels of constant light mimics aspects of human retinal degeneration. With collaborators we conducted gene expression profile experiments of rat retina exposed to bright light and identified upregulation of 35 genes and downregulation of 30 other genes. We are evaluating associated biological processes including oxidative stress, cellular defenses, DNA damage and cell death, and carbohydrate and lipid metabolism. Rab38cht/cht mice: The spontaneous coat color mutant, chocolate (cht), is caused by homozygous mutation (G146T->Gly19Val) in the small GTP-binding protein, rab38. cht mice are a model for Hermansky-Pudlak-like syndromes and may be useful to study of ocular melanosome biogenesis Clinical iris and retinal pigment epithelium (RPE) atrophy develops in approximately 30% and 70%, respectively, of homozygous mice (n = 31). We screened these mice for functional deficits using the electroretinogram. Responses in 3 month old rab38cht/cht mice showed a slightly larger amplitudes compared to rab38cht/+ mice and may reflect differences in malanosome content or distribution. RanBP2+/- mice : RanBP2 is a large scaffold protein unique to the vertebrate photoreceptors inner segment where it interacts with proteins mitochondrial. RanBP2 is involved in human choroideremia. The ERG of heterozygous knockout mice is reduced and is the first instance of a heterozygous mutation in an inner segment protein having an effect on the photoreceptor light response. Origins of the Photopic Negative Response (PhNR) in Retinal Degeneration in the Rat: The PhNR in RCS rats may originate with potassium currents in the inner retina. This response become more prominent in some rodent models of retinal degeneration but not in others. Investigation of this phenomenon may lead to greater understanding of the effects of photoreceptor degeneration on the post-photoreceptoral pathways. We investigated the distribution and level of Kir4.1, the dominant potassium channel on Mueller glia cells responsible for generating the ERG currents in the inner retina, in two rodent models of retinal degeneration. Though both forms of retinal degeneration are characterized by photoreceptor death, the photopic ERGs and the PhNR demonstrate that they involve a different set of events. Retinal pathways giving rise to the ERG phenotype in human congenital stationary night blindness (CSNB1-NYX). These data were collected in our lab at the University of Michigan but analyzed and written here. Retinal ON-pathway dysfunction is implicated in human CSNB1. We probed cone pathway dysfunction in four human genotyped CSNB1 affected males by ERG recordings with photopic sinusoidal and rapid-on/-off ramp flicker stimuli that elicit ON-/OFF-pathway activity selectively. We created the human ERG abnormalities created in anesthetized non-human primates by intravitreal application of glutamate analogs that selectively suppress retinal ON- or OFF-pathway bipolar cell activity. The effects of ciliary neurotrophic factor (CNTF) on retinal function of the normal rabbit. Bolus CNTF injection can protect against photoreceptor cell loss in rodent retinal degeneration models, but chronic delivery by gene transfer impaired retinal function even though CNTF substantially preserved photoreceptor numbers. We investigated ERG and histological changes in normal rabbits following intravitreal implants of encapsulated cell technology (ECT) devices containing human RPE releasing CNTF. We currently are testing CNTF in humans (see protocols below). ECT-CNTF at therapeutic doses did not impair rabbit rod or cone ERG function, although dose-related photoreceptor nuclei changes were seen. Single large bolus CNTF injections into the rat eye strongly suppressed the ERG response within one week but recovered within three weeks, correlated with suppression and then reversal of phototransduction protein expression and photoreceptive membrane, indicating a mechanism for ERG suppression seen previously. Human Protocol 03-EI-0033. X-Linked Juvenile Retinoschisis - Clinical and Molecular Studies. (PI: P.A. Sieving). A genotype-phenotype study of XLRS which results in splitting of the retinal layers. A better understanding of XLRS development might lead to improved treatments through gene transfer. Human Protocol 03-EI-0179. Investigation of the Effect of Dietary Docosahexaenoic Acid (DHA) Supplementation on Macular Function in Subjects with Autosomal Dominant Stargardt-Like and Autosomal Recessive Stargardt Macular Dystrophy. (PI: P.A. Sieving). Evaluate DHA supplementation to improve macular function in patients with Stargardt and Stargardt-like macular dystrophies. DHA fatty acid is essential for normal brain and eye development. Human Protocol 03-EI-0234. A Phase I Study of NT-501-10 and NT-501-6A.02, Implants of Encapsulated Human NTC-210 Cells Releasing Ciliary Neurotrophic Factor (CNTF), in Patients with Retinitis Pigmentosa. (PI: P.A. Sieving). Evaluate safety of CNTF implant in the human eye of retinal degeneration subjects. CNTF protects against retinal degeneration in animal models. Study addresses a major treatment challenge of delivery directly into the human eye. Human Protocol 03-EI-0255. Pilot Study on the Effect of Vitamin A Supplementation on Cone Function in Retinitis Pigmentosa (RP). (PI: P.A. Sieving). A protocol to evaluate whether high dose oral vitamin A will improve retinal function acutely in patients with RP.